Process for producing arsenic trioxide formulations and methods for treating cancer using arsenic trioxide or melarsoprol

ABSTRACT

The invention relates to the use of arsenic compounds to treat a variety of leukemia, lymphoma and solid tumors. Further, the arsenic compounds may be used in combination with other therapeutic agents, such as a retinoid. The invention also provides a process for producing arsenic trioxide formulations.

FIELD OF INVENTION

[0001] The present invention relates to methods and compositions for thetreatment of leukemia, lymphoma, and certain other cancers.

[0002] More specifically, the present invention relates to the noveluses of arsenic trioxide and an organic arsenic compound for treatingacute leukemia and chronic leukemia.

BACKGROUND OF THE INVENTION

[0003] Cancer

[0004] Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

[0005] Pre-malignant abnormal cell growth as exemplified by hyperplasia,metaplasia, and dysplasia (for review of such abnormal growthconditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B.Saunders Co., Philadelphia, pp. 68-79) precedes the formation of aneoplastic lesion. A neoplastic lesion may evolve clonally to grow intoa solid tumor, and develop an increasing capacity for invasion, growth,metastasis, and heterogeneity, especially under conditions in which theneoplastic cells escape the host's immune surveillance (Roitt, I.,Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis,pps. 17.1-17.12).

[0006] Leukemia refers to malignant neoplasms of the blood-formingtissues. Transformation to malignancy typically occurs in a single cellthrough two or more steps with subsequent proliferation and clonalexpansion. In some leukemias, specific chromosomal translocations havebeen identified with consistent leukemic cell morphology and specialclinical features (e.g., translocations of 9 and 22 in chronicmyelocytic leukemia, and of 15 and 17 in acute promyelocytic leukemia).Acute leukemias are predominantly undifferentiated cell populations andchronic leukemias more mature cell forms.

[0007] Acute leukemias are divided into lymphoblastic (ALL) andnon-lymphoblastic (ANLL) types. They may be further subdivided by theirmorphologic and cytochemical appearance according to theFrench-American-British (FAB) classification or according to their typeand degree of differentiation. The use of specific B- and T-cell andmyeloid-antigen monoclonal antibodies are most helpful forclassification. ALL is predominantly a childhood disease which isestablished by laboratory findings and bone marrow examination. ANLL,also known as acute myeloblastic leukemia (AML), occurs at all ages andis the more common acute leukemia among adults; it is the form usuallyassociated with irradiation as a causative agent.

[0008] Chronic leukemias are described as being lymphocytic (CLL) ormyelocytic (CML). CLL is characterized by the appearance of maturelymphocytes in blood, bone marrow, and lymphoid organs. The hallmark ofCLL is sustained, absolute lymphocytosis (>5,000/μL) and an increase oflymphocytes in the bone marrow. Most CLL patients also have clonalexpansion of lymphocytes with B-cell characteristics. CLL is a diseaseof older persons. In CML, the characteristic feature is the predominanceof granulocytic cells of all stages of differentiation in blood, bonemarrow, liver, spleen, and other organs. In the symptomatic patient atdiagnosis the total WBC count is usually about 200,000/μL, but may reach1,000,000/μL. CML is relatively easy to diagnose because of the presenceof the Philadelphia chromosome.

[0009] The very nature of hematopoietic cancer necessitates usingsystemic chemotherapy as the primary treatment modality. Drugs selectedaccording to sensitivities of specific leukemias are usually given incombination. Radiation therapy may be used as an adjunct to treat localaccumulations of leukemic cells. Surgery is rarely indicated as aprimary treatment modality, but may be used in managing somecomplications. Bone marrow transplantation from an HLA-matched siblingis sometimes indicated.

[0010] Arsenic and its Medical Uses

[0011] Arsenic has been considered to be both a poison and a drug for along time in both Western and Chinese medical practices. In the latterpart of the nineteenth century, arsenic was used frequently in attemptsto treat diseases of the blood in the West. In 1878, it was reportedthat treatment of a leukemic patient with Fowler's solution (a solutioncontaining potassium arsenite, valence +5) reduced markedly the count ofwhite blood cells (Cutler and Bradford, Am. J. Ned. Sci., January 1878,81-84). Further interests in the use of Fowler's solution as apalliative agent to treat chronic myelogenous leukemia (CML) wasdescribed by Forkner and Scott in 1931 (J. Am. Med. Assoc., 1931, iii,97), and later confirmed by Stephens and Lawrence in 1936 (Ann. Intern.Med. 9, 1488-1502). However, while the active chemical ingredient(s) ofFowler's solution was not determined, its toxicity was well recognized.Fowler's solution was administered strictly as an oral composition, andwas given to leukemic patients as a solution until the level of whiteblood cells was depressed to an acceptable level or until toxicities(such as skin keratoses and hyperpigmentation) developed, while thepatients enjoyed varying periods of remission. In the 1960's, Fowler'ssolution was still used occasionally in attempts to treat CML, however,most patients with CML were treated with other chemotherapeutic agents,such as busulfan, and/or radiation therapy (Monfardini et al., Cancer,1973, 31:492-501).

[0012] Paradoxically, one of the long recognized effects of exposure toarsenic, whether the source is environmental or medicinal, is skincancer (Hutchinson, 1888, Trans. Path. Soc. Lond., 39:352; Neubauer,1947, Br. J. Cancer, 1:192). There were even epidemiological data tosuggest that the use of Fowler's solution over long periods could leadto an increased incidence of cancer at internal sites (Cuzick et al.,Br. J. Cancer, 1982, 45:904-911; Kaspar et al., J. Am. Med. Assoc.,1984, 252:3407-3408). The carcinogenicity of arsenic has since beendemonstrated by the fact that it can induce chromosomal aberration, geneamplification, sister chromatid exchanges and cellular transformation(See e.g., Lee et al., 1988, Science, 241:79-81; and Germolec et al.,Toxicol. Applied Pharmacol., 1996, 141:308-318). Because of the knowncarcinogenic effect of arsenic, its only therapeutic use in human inWestern medicine today is in the treatment of tropical diseases, such asAfrican trypanosomiasis, (the organic arsenical, melarsoprol; SeeGoodman & Gilman's The Pharmacological Basis of Therapeutics, 9thedition, chapter 66, 1659-1662, 1997).

[0013] In traditional chinese medicine, arsenous acid or arsenictrioxide paste has been used to treat tooth marrow diseases, psoriasis,syphilis and rheumatosis (Chen et al., 1995, in Manual of ClinicalDrugs, Shanghai, China, Shanghai Institute of Science and Technology,p.830). In 1970's, arsenic trioxide had been applied experimentally totreat acute promyelocytic leukemia (APL) in China (commented by Mervis,1996, Science, 273:578). The clinical efficacy of arsenic trioxide hasrecently been re-investigated in 14 of 15 patients with refractory APL,where the use of an intravenous dose at 10 mg/day for 4-9 weeks wasreported to result in complete morphologic remission without associatedbone marrow suppression (Shen et al., 1997, Blood, 89:3354-3360). It wasalso shown that arsenic trioxide induced apoptosis (programmed celldeath) in vitro in NB4 cells, an APL cell line, and that apoptosis wasapparently associated with down-regulation of the oncogene bcl-2, andintracellular redistribution of the chimeric PML/RARα protein that areunique to APL cells (Chen et al., 1996, Blood, 88:1052-1061; Andre etal., 1996, Exp. Cell Res. 229:253-260). It has been reported that thebiological activity of arsenic is due to the ability of arsenic todirect the nucleoplasmic fraction of PML to nuclear bodies fordegradation (Zhu et al., 1997, Proc. Natl. Acad. Sci., 94:3978-3983).

[0014] Although arsenic is well known to be both a poison and acarcinogenic agent, there have been many reports concerning the use ofarsenic in medical treatment. Further, from the above discussion, itshould be clear that there are many different types of leukemias, eachof which requires a unique treatment protocol that is modified accordingto the presence of factors predicting for a risk of treatment failure.Thus, the development of a broad spectrum anti-leukemia agent that canbe used alone or in combination with other existing drugs is extremelydesirable.

SUMMARY OF THE INVENTION

[0015] Despite the conflicting reports in the art concerning benefitsand risks of the administration of arsenic to patients, applicants havediscovered that arsenic trioxide and the organic arsenical, melarsoprol,have broad applicability in the treatment of various types of leukemias,lymphomas, and solid tumors.

[0016] The invention described herein encompasses a method of treatingleukemia, lymphoma or solid tumors comprising the administration of atherapeutically effective and non-lethal amount of arsenic trioxide ormelarsoprol to a human in need of such therapy. The invention, asmentioned above also encompasses the use of combination therapy to treatleukemia, especially leukemias which are refractory to other forms oftreatment.

[0017] The invention also encompasses a method for the manufacture ofpharmaceutical compositions comprising arsenic trioxide.

[0018] In accordance with the present invention, arsenic trioxide ormelarsoprol compounds can be used alone or in combination with otherknown therapeutic agents (including chemotherapeutics, radioprotectantsand radiotherapeutics) or techniques to either improve the quality oflife of the patient, or to treat leukemia, lymphoma or solid tumor. Thearsenic compounds can be used before, during or after the administrationof one or more known chemotherapeutic agents, including antitumoragents. In addition, the arsenic compounds can be used before, during orafter radiation treatment.

[0019] The pharmaceutical compositions of the invention are sterilesolutions suitable for intravenous injection or infusion. In anotherembodiment the invention encompasses a composition suitable for oraldelivery; comprising arsenic trioxide or melarsoprol and apharmaceutically acceptable excipient or carrier. In another embodiment,the invention also includes compositions suitable for topical ortransdermal delivery, including but not limited to iontophoreticmethods. Specific therapeutic regimens, pharmaceutical compositions, andkits are also provided by the invention.

[0020] Particular compositions of the invention and their uses aredescribed in the sections and subsections which follow.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Methods and compositions for the treatment of leukemia, lymphomaor solid tumors are described herein. This invention provides a methodof treating acute or chronic leukemia, lymphoma, or solid tumors in ahuman which comprises administering to a human in need of such therapy atherapeutically effective and non-lethal amount of one or more arseniccompounds, such as arsenic trioxide or melarsoprol.

[0022] The invention also includes a method of treating leukemia in ahuman who has become refractory to other forms of treatment whichcomprises administering to a human arsenic trioxide or melarsoprol incombination with another chemotherapeutic agent, e.g., all-transretinoic acid (ATRA).

[0023] The invention also relates to a method for the manufacture ofpharmaceutical compositions comprising arsenic trioxide. It is preferredthat pharmaceutical compositions of the present invention exhibitreduced toxicity, improved efficacy, improved stability during storageand use, and that the composition has a physiologically acceptable pH.

[0024] The Arsenic Compounds

[0025] As used herein, “arsenic compound” refers to a pharmaceuticallyacceptable form of arsenic trioxide (As₂O₃) or melarsoprol. Melarsoprolis an organic arsenic compound which can be synthesized by complexingmelarsen oxide with dimercaprol or commercially purchased (Arsobal® byRhone Poulenc Rorer, Collegeville, Pa.). Since the non-pharmaceuticallyformulated raw materials of the invention are well known, they can beprepared from well-known chemical techniques in the art. (See forexample, Kirk-Othmer, Encyclopedia of Chemical Technology 4th ed. volume3 pps. 633-655 John Wiley & Sons).

[0026] As used herein the terms “a therapeutic agent”, “therapeuticregimen”, “radioprotectant”, “chemotherapeutic” mean conventional drugsand drug therapies, including vaccines, for treating cancer, viralinfections, and other malignancies, which are known to those skilled inthe art. “Radiotherapeutic” agents are well known in the art.

[0027] In accordance with the present invention, arsenic trioxide ormelarsoprol compounds can be used alone or in combination with otherknown therapeutic agents (including chemotherapeutics, radioprotectantsand radiotherapeutics) or techniques to either improve the quality oflife of the patient, or to treat leukemia, lymphoma or solid tumor. Forexample, the arsenic compounds can be used before, during or after theadministration of one or more known antitumor agents including but notlimited to mustard compounds, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, floxuridine, methotrexate, vincristine, vinblastine,taxol, etoposide, temiposide, dactinomycin, daunorubicin, doxorubicin,bleomycin, mitomycin, cisplatin, carboplatin, estramustine phosphate,hydroxyurea, BCNU, procarbazine, VM-26, interferons, and all-transretinoic acid (ATRA), or other retinoids (See, for example, thePhysician Desk References 1997). In addition, the arsenic compounds canbe used before, during or after radiation treatment.

[0028] In a specific embodiment, the arsenic compound of the inventionand ATRA can be administered as a mixture. In preferred aspects, thelymphoma, leukemia or solid tumor in the human treated by thecombination is refractory to general methods of treatment, or is arelapsed case of leukemia.

[0029] Any suitable mode of administration may be used in accordancewith the present invention including but not limited to parenteraladministration such as intravenous, subcutaneous, intramuscular andintrathecal administration; oral, and intranasal administration, andinhalation. The mode of administration will vary according to the typeof cancer, and the condition of the human.

[0030] The pharmaceutical compositions to be used may be in the form ofsterile aqueous or organic solutions; colloidal suspensions, caplets,tablets and cachets.

[0031] Methods Of Treatment

[0032] The term “a method for treating leukemia” as used herein meansthat the disease and the symptoms associated with the disease arealleviated, reduced, cured, or placed in a state of remission. Forexample, the methods of treatment of the invention can lower the whiteblood cell count, or reduce lymphocytosis in a human under treatment.

[0033] The term “a method for treating lymphoma” as used herein meansthat the disease and the symptoms associated with the disease arealleviated, reduced, cured, or placed in a state of remission.

[0034] The term “a method for treating solid tumor” as used herein meansthat the disease and the symptoms associated with the solid tumor arealleviated, reduced, cured, or placed in a state of remission.

[0035] In addition, the term “a method for treating leukemicinfiltration” means that the infiltration of leukemic cells out ofcirculation and into other organs and systems and the symptomsassociated with such infiltration are alleviated, reduced, cured, orplaced in a state of remission.

[0036] The term “refractory” when used herein means that the leukemia isgenerally resistant to treatment or cure.

[0037] As used herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; or cells that fail todifferentiate normally; and morphological evidence, increasinglysupported by molecular biologic studies, indicates that preneoplasiaprogresses through multiple steps.

[0038] In one embodiment, the invention provides a method for treatmentof leukemia in a human comprising the administration of atherapeutically effective and non-lethal amount of arsenic trioxide ormelarsoprol to the human. The invention also provides a weight-baseddosing regimen, not heretofore disclosed, that maximizes the safety inhumans of these otherwise highly toxic compounds.

[0039] Arsenic trioxide (As₂O₃) inhibits growth and induce apoptosis inNB4 acute promyelocytic leukemic cells. Acute promyelocytic leukemia(APL) is associated with the t(15;17) translocation, which generates aPML/RARα fusion protein between PML, a growth suppressor localized onnuclear matrix-associated bodies, and RARα, a nuclear receptor forretinoic acid (RA). PML/RARα was proposed to block myeloiddifferentiation through inhibition of nuclear receptor response, as doesa dominant negative RARα mutant. In addition, in APL cells, PML/RARαdisplaces PML and other nuclear body (NB) antigens onto nuclearmicrospeckles, likely resulting in the loss of PML and/or NB functions.It has been suggested that high concentrations of arsenic trioxidepromote apoptosis, whereas low concentrations induce partialdifferentiation in NB4 cells as well as cells derived from APL patients.It was postulated that As₂O₃ works through its ability to specificallycause PML-RARα in APL cells to be relocalized to nuclear bodies fordegradation (Zhu et al., 1997, Proc. Natl. Acad. Sci. USA,94:3978-3983). However, these findings tend to limit the use of arsenictrioxide to a subset of leukemias. See Konig et al., 1997, Blood,90:562-570.

[0040] Unexpectedly, the inventors have discovered that both As₂O₃ andmelarsoprol are able to inhibit cell growth, and induce apoptosis invarious myeloid leukemia cell lines in a PML and PML-RARα independentmanner. Thus, the inventors have discovered that, contrary to theearlier findings, arsenic trioxide. and melarsoprol are both effectiveagainst a broad range of leukemias regardless of the underlyingmolecular mechanism that causes the neoplasia. Working examples of theeffect of arsenic compounds on a number of leukemic cell lines areprovided in Sections 5.1 and 5.2.

[0041] Accordingly, the arsenic compounds of the present invention canbe used against a variety of leukemias, including but not limited to;

[0042] Acute lymphoblastic leukemia (ALL)

[0043] Acute lymphoblastic B-cell leukemia

[0044] Acute lymphoblastic T-cell leukemia

[0045] Acute myeloblastic leukemia (AML)

[0046] Acute promyelocytic leukemia (APL)

[0047] Acute monoblastic leukemia

[0048] Acute erythroleukemic leukemia

[0049] Acute megakaryoblastic leukemia

[0050] Acute myelomonocytic leukemia

[0051] Acute undifferentiated leukemia

[0052] Chronic myelocytic leukemia (CML)

[0053] Chronic lymphocytic leukemia (CLL)

[0054] The skilled artisan will recognize that other leukemias may betreated in accordance with the present invention.

[0055] In another embodiment, the invention provides a method fortreatment of lymphoma in a human comprising the administration of atherapeutically effective and non-lethal amount of arsenic trioxide ormelarsoprol to the human. Lymphoma that can be treated by the methods ofthe invention include but are not limited to high grade lymphoma,intermediate grade lymphoma, low grade lymphoma, and the varioussubclassifications.

[0056] In yet another embodiment, the invention provides a method fortreatment of solid tumors, including metastasises, in humans comprisingthe administration of a therapeutically effective and non-lethal amountof arsenic trioxide or melarsoprol to the human. Solid tumors that canbe treated by the methods of the invention include but are not limitedto: cancer of the digestive tract, oesophagus, liver, stomach, andcolon; skin; brain; bone; breast; lung; and soft tissues, including butnot limited to various sarcomas, and preferably prostate cancer.

[0057] In various embodiments, the leukemic or tumor cells areinfiltrating other organs and systems in a human, for example, thecentral nervous system. The methods of the invention are also applicableto reduce the number of preneoplastic cells in a human in which there isan abnormal increase in the number of preneoplastic cells.

[0058] In a specific embodiment, the invention provides a method oftreatment of acute promyelolytic leukemia (APL) in a human comprisingthe administration of a therapeutically effective and non-lethal amountof melarsoprol to the human. The inventors discovered, as described inSection 5.2, that concentrations of melarsoprol that are cytotoxic invitro can readily be achieved in vivo.

[0059] In one specific embodiment, the invention provides a method oftreatment of chronic myelogenous leukemia (CML) in a human comprisingthe administration of a therapeutically effective and non-lethal amountof arsenic trioxide to the human. The inventors discovered, as describedin Section 5.3, that arsenic trioxide can also induce apoptosis in a CMLcell line. The therapeutic benefits of the pharmaceutical compositionsof the invention comprising arsenic trioxide is far superior to that ofpotassium arsenite, commonly formulated as Fowler's solution.

[0060] In yet another specific embodiment, the invention provides amethod of treatment of acute promyelocytic leukemia (APL) in a human, inwhich the APL is associated with a translocation of the RARα locus onchromosome 17 to chromosome 11, comprising the administration of atherapeutically effective amount of arsenic trioxide or melarsoprol tothe human. In the majority of APL cases, RARα on chromosome 17translocates and fuses with the PML gene located on-chromosome 15, i.e.,t(15;17). In a few cases RARA translocates to chromosome 11 where itfuses to the PLZF gene. Patients harboring the t(15;17) are uniquelysensitive to treatment with all-trans retinoic acid (ATRA), yieldingcomplete remission rates of 75% to 95%. APL associated with the t(11;17)(PLZF-RARa) shows a distinctly worse prognosis with poor response tochemotherapy and little or no response to treatment with ATRA, thusdefining a new APL syndrome. The present invention provides that arsenictrioxide or melarsoprol can be used to treat such cases of APL.Transgenic animal models of APL associated with t(15;17) and t(11;17)for testing the therapeutic benefits and dosages of arsenic compounds ofthe invention are described in Section 5.4 hereinbelow.

[0061] Humans having leukemia are sometimes refractory to conventionalmethods of treatment by reason of having undergone anti-leukemic therapy(e.g., chemotherapy). Thus, the invention provides a method of treatmentof leukemia in a human who is not responding to conventional therapycomprising the administration of a therapeutically effective andnon-lethal amount of a combination of arsenic compound and anotherchemotherapeutic agent, such as but not limited to, all-trans retinoicacid (ATRA) or other retinoids, to the human. The arsenic compound caneither be arsenic trioxide or melarsoprol or a pharmaceuticallyacceptable salt thereof. The invention also encompasses the treatment ofretinoid-resistant patients with an arsenic compound.

[0062] In specific embodiments, the arsenic compound of the inventionand the chemotherapeutic agent can be administered either as a mixtureor sequentially. When administered sequentially, the arsenic compoundmay be administered before or after the chemotherapeutic agent, so longas the first administered agent is still providing antileukemic activityin the human when the second agent is administered. Any of the modes ofadministration described herein may be used to deliver the combination.In preferred aspects, the leukemia in the human treated by thecombination is refractory to general methods of treatment, or is arelapsed case of leukemia.

[0063] Process for The Manufacture Of Sterile Arsenic Trioxide Solution

[0064] The arsenic compounds of the invention may be formulated intosterile pharmaceutical preparations for administration to humans fortreatment of leukemias, lymphomas and solid tumors. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may be prepared, packaged, labelled for treatmentof and used for the treatment of the indicated leukemia, lymphoma, orsolid tumor.

[0065] In one aspect, the invention provides a method for themanufacture of a pharmaceutical composition comprising a therapeuticeffective and non-lethal amount of arsenic trioxide (As₂O₃). Arsenictrioxide (raw material) is a solid inorganic compound that iscommercially available in a very pure form. However, it is difficult todissolve As₂O₃ in aqueous solution. Further, the inventors are unawareof any published teachings on how to formulate As₂O₃ as a pharmaceuticalcomposition suitable for injection directly into a human. Arsenic is.present in solution in the +5 valence state (pentavalent) or the +3valence state (trivalent). For example, potassium arsenite (KAsO₂; whichis present in Fowler's solution) and salts of arsenious acid containpentavalent arsenic. It is known that one form of arsenic is more toxicthan the other. (Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th edition, chapter 66, 1660, 1997). A fresh solution ofarsenic trioxide containing arsenic in the trivalent state will begradually oxidized to pentavalent state if exposed to air for aprolonged period, and as a result of the accumulation of pentavalentarsenic, the relative toxicity of a solution of As₂O₃ will change overtime. (Id.) Furthermore, it is observed that the total amount of arsenicin solution decreases over time. This loss of material is caused by theprogressive conversion of arsenic in the solution into arsine (AsH₃)which is a gaseous compound at room temperature. This is particularlyproblematic in pharmaceutical applications if the concentration of anactive ingredient in the injected material cannot be controlled. It isalso undesirable to allow arsine to escape from the solution into theatmosphere because arsine is also toxic.

[0066] The inventors have experimented and successfully developed amethod for formulating arsenic trioxide which overcomes theabove-described problems of solubility and stability. The methodcomprises solubilizing solid high purity As₂O₃ in an aqueous solution athigh pH, such as pH greater than 12. For example, a 5 M solution ofsodium hydroxide may be used. To aid solubilization and obtain a clearand homogenous solution, mechanical stirring and/or gentle heating maybe applied. A solution of As₂O₃ can also be obtained by dissolving thesolid compound overnight. Typically, a solution of 1 M As₂O₃ is obtainedby this method. However, this solution is too basic to be useful as apharmaceutical composition.

[0067] To adjust the pH of the As₂O₃ solution, the solution is firstdiluted in water, for example, to a concentration of about 1 mg/mL, pH12. The As₂O₃ solution is then back-titrated with acid, such as,hydrochloric acid (1 M to 5 M HC1), with constant stirring until the pHis about 8.0 to 8.5. Highly concentrated HCl is not suitable as itcauses precipitation to occur in the As₂O₃ solution. The partiallyneutralized As₂O₃ solution may then be sterilized for example, byfiltration (e.g., through a 0.22 μm filter), and stored in sterilevials.

[0068] To make a pharmaceutical composition that can be directlyinjected into a subject, the composition must be sterile, standardtechniques known to the skilled artisan for sterilization can be used.See, e.g., Remington's Pharmaceutical Science. The pH of the partiallyneutralized As₂O₃ solution may be further adjusted to near physiologicalpH by dilution (10-100 fold) with a pharmaceutical carrier, such as a 5%dextrose solution. For example, 10 mL of a partially neutralized As₂O₃solution can be added to 500 mL of a 5% dextrose solution to yield afinal pH of about 6.5 to 7.5. The method of the invention reduces theoxidation of arsenic in solution. Pharmaceutical compositions containingarsenic trioxide manufactured by the method of the invention showimproved stability and long shelf life.

[0069] Pharmaceutical Composition and Modes Of Administration

[0070] According to the invention, the arsenic compounds and theirphysiologically acceptable solvates may be formulated for oral orparenteral administration.

[0071] For oral administration, the pharmaceutical preparation may be inliquid form, for example, solutions, syrups or suspensions, or may bepresented as a drug product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

[0072] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0073] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or is continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0074] The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of the arsenic compounds inpharmaceutically acceptable form. The arsenic compound in a vial of akit of the invention may be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the complex may be lyophilized or desiccated; inthis instance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

[0075] In another embodiment, a kit of the invention further comprises aneedle or syringe, preferably packaged in sterile form, for injectingthe complex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of arsenic compounds by a clinician or bythe patient.

[0076] The magnitude of a therapeutic dose of an arsenic compound in theacute or chronic management of leukemia will vary with the severity ofthe condition to be treated and the route of administration. The dose,and perhaps dose frequency, will also vary according to the age, bodyweight, condition and response of the individual patient. In general,the daily dose ranges of arsenic trioxide for the conditions describedherein are generally from about 0.05 to about 5 mg per kg body weightadministered in divided doses administered parenterally or orally ortopically. A preferred total daily dose is from about 2.5 to about 40 mgof arsenic trioxide. Preferably the arsenic trioxide formulation of theinvention is given daily for a maximum of 60 days, or until remission,followed by two to ten additional cycles, each lasting about 25 days induration. For example, depending on the body weight of a patient withacute promyelocytic leukemia, a daily dose of arsenic trioxide greaterthan or less than 10 mg can be administered. Alternatively, followingthe weight-based dosing regimen, a therapeutic effect can be obtainedwith a daily dose of arsenic trioxide less than 10 mg.

[0077] For treatment of solid tumor, a preferred dosing regimen involvesintravenous infusion of about 0.1 to about 5 mg per kg body weight perday for 5 days. This five-day treatment protocol is repeated once permonth until the tumor growth tumor is inhibited or when the tumor showssigns of regression.

[0078] As for melarsoprol, the total daily dose ranges for theconditions described herein are generally from about 0.1 to about 5mg/kg body weight administered in divided doses administeredparenterally or orally or topically. A preferred total daily dose isfrom about 0.5 to about 4 mg melarsoprol per kg body weight.

[0079] The effect of the therapy with arsenic trioxide or melarsoprol ondevelopment and progression of cancer can be monitored by any methodsknown in the art, including but not limited to determining: levels oftumor specific antigens and putative biomarkers, _e.g., carcinoembryonicantigens (CEA), alpha-fetoprotein; and changes in morphology and/or sizeusing computed tomographic scan and/or sonogram.

[0080] Desirable blood levels may be maintained by a continuous infusionof an arsenic compound as ascertained by plasma levels. It should benoted that the attending physician would know how to and when toterminate, interrupt or adjust therapy to lower dosage due to toxicity,or bone marrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

[0081] Again, any suitable route of administration may be employed forproviding the patient with an effective dosage of an arsenic compound.For example, oral, transdermal, iontophoretic, parenteral (subcutaneous,intramuscular, intrathecal and the like) may be employed. Dosage formsinclude tablets, troches, cachet, dispersions, suspensions, solutions,capsules, patches, and the like. (See, Remington's PharmaceuticalSciences.)

[0082] The pharmaceutical compositions of the present invention comprisean arsenic compound as the active ingredient, pharmaceuticallyacceptable salt thereof, and may also contain a pharmaceuticallyacceptable carrier, and optionally, other therapeutic ingredients, forexample all trans retinoic acid. The term “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic acids and bases, including inorganic and organic acids andbases.

[0083] The pharmaceutical compositions include compositions suitable fororal, mucosal routes, transdermal, iontophoretic, parenteral (includingsubcutaneous, intramuscular, intrathecal and intravenous), although themost suitable route in any given case will depend on the nature andseverity of the condition being treated.

[0084] In the case where an intravenous injection or infusioncomposition is employed, a suitable dosage range for use is, e.g., fromabout one to about 40 mg arsenic trioxide total daily; about 0.001 toabout 10 mg arsenic trioxide per kg body weight total daily, or about0.1 to about 10 mg melarsoprol per kg body weight total daily.

[0085] In addition, the arsenic carrier could be delivered via chargedand uncharged matrices used as drug delivery devices such as celluloseacetate membranes, also through targeted delivery systems such asfusogenic liposomes attached to antibodies or specific antigens.

[0086] In practical use, an arsenic compound can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including tablets, capsules, powders, intravenous injections orinfusions). In preparing the compositions for oral dosage form any ofthe usual pharmaceutical media may be employed, e.g., water, glycols,oils, alcohols, flavoring agents, preservatives, coloring agents, andthe like; in the case of oral liquid preparations, e.g., suspensions,solutions, elixirs, liposomes and aerosols; starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like in the case of oral solidpreparations e.g., powders, capsules, and tablets. In preparing thecompositions for parenteral dosage form, such as intravenous injectionor infusion, similar pharmaceutical media may be employed, e.g., water,glycols, oils, buffers, sugar, preservatives and the like know to thoseskilled in the art. Examples of such parenteral compositions include,but are not limited to Dextrose 5% w/v, normal saline or othersolutions. The total dose of the arsenic compound may be administered ina vial of intravenous fluid, e.g., ranging from about 2 ml to about 2000ml. The volume of dilution fluid will vary according to the total doseadministered. For example, arsenic trioxide supplied as a 10 ml aqueoussolution at 1 mg/ml concentration is diluted in 10 to 500 ml of 5%dextrose solution, and used for intravenous infusion over a period oftime ranging from about ten minutes to about four hours.

[0087] An exemplary course of treatment of a patient with leukemia,lymphoma, or solid cancer can involve daily administration byintravenous infusion of arsenic trioxide in an aqueous solution at adaily dose of about 0.01 to 1 mg arsenic trioxide per kg of body weightof the patient. Preferably, about 0.15 mg arsenic trioxide per kg bodyweight per day is used. The course of treatment may continue until bonemarrow remission is observed or when side effects are becoming serious.The course of treatment may be repeated for up to ten times overapproximately 10 months with a break of about 3 to 6 weeks in betweencourses. The post-remission course of treatment involves infusion ofarsenic trioxide at a daily dose of about 0.15 mg per kg of body weightof the patient on a daily or weekdays-only basis for a cumulative totalof 25 days.

EXAMPLES

[0088] Described below are examples of uses of the arsenic compounds ofthe invention in treatment of various types of leukemia. Through theseand other experiments the arsenic trioxide formulation of the inventionwere found to be well-tolerated in humans. For example, three APLpatients were given 10 mg of the arsenic trioxide formulation of theinvention once daily (flat dose) intravenous dose.

[0089] Arsenic Trioxide and Melarsoprol Induce Apoptosis in MyeloidLeukemia Cell Lines

[0090] The activity of As₂O₃ and melarsoprol against myeloid leukemiacell lines, including the APL cell line NB4-306 (a retinoicacid-resistant cell line derived from NB4 that no longer expresses theintact PML-PARα fusion protein), HL60, KG-1, and the myelomonocytic cellline U937 was investigated. To examine the role of PML in mediatingarsenical activity, the inventors also tested these agents using murineembryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in whichthe PML gene had been inactivated by homologous recombination.Unexpectedly, it is found that both compounds inhibited cell growth andinduced apoptosis in all cell lines tested. Melarsoprol was more potentthan As₂O₃ at equimolar concentrations ranging from 10⁻⁷ to 10⁻⁵ mol/L.As₂O₃ relocalized PML and PML-RARα onto nuclear bodies, which wasfollowed by PML degradation in NB4 as well as in HL60 and U937 celllines. Although melarsoprol was more potent in inhibiting growth andinducing apoptosis, it did not affect PML and/or PML-RARα nuclearlocalization. Moreover, both As₂O₃ and melarsoprol comparably inhibitedgrowth and induced apoptosis of PML+/+ and PML−/−MEF, and inhibitedcolony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyteformation in BM cultures of PML+/+ and PML−/− progenitors. A detaileddescription of the methods, materials, and results of these experimentsis provided in Wang et al., Blood, 1998, 92:1497-1504.

[0091] Results from the experiments show that the cytotoxic effect ofboth arsenicals in these cell lines is not mediated by mechanisms thatare dependent on PML or PML-RAR+ expression. In most lines, melarsoprolwas somewhat more potent compared with As₂O₃ in inhibiting growth andinducing apoptosis, and the effects of both drugs were dose dependent.As previously reported, it is confirmed that As₂O₃ relocalized PMLprotein onto nuclear bodies and induced PML and PML-RARα degradation inNB4 cells while triggering spoptosis. However, similar effects were alsoobserved in HL60 and U937 cells which do not harbor the PML-RARα fusiongene. Moreover, melarsoprol induced apoptosis in all the cell linestested without altering PML and/or PML-RARα.

[0092] The differentiating action of As₂O₃ and melarsoprol, appearednegligible in vitro, and did not appear to depend on the expressionand/or modulation of PML and/or PML-RARα either. In fact, the smalleffect observed by the inventors in long-term cultures (up to 2 weeks),was comparable in all the cell lines tested with both compounds.

[0093] It is also found that bcl-2 downregulation, which has beenpreviously linked to the antileukemic effects of As₂O₃ in APL, was alsonot dependent on expression of PML-RARα protein, because it occurred inthe NB4 subclone 306 in which the intact protein is not detectable.Finally, to test whether PML expression was essential to theantileukemic effects of arsenicals, both agents were tested in mouseembryonic fibroblasts and BM cells from animals wherein wild-type PMLhad been eliminated by homologous recombination. In these cells whollylacking PML expression, both As₂O₃ and melarsoprol were equallyeffective in inhibiting growth and inducing apoptosis, and both hadsimilar effects on normal CFU-E and CFU-GM colony formation. Moreover,no differences between wild-type and PML−/− cells were observed. Withoutbeing limited by any theory, together, these data strongly supporttheory that the antileukemic effects of these arsenicals occursindependently of both PML and PML-RARα expression. These results are inkeeping with the medicinal history of arsenicals for diseases that arenot characterized by alterations in PML protein such as, for instance,chronic myelocytic leukemia.

[0094] The results indicate that both As₂O₃ and melarsoprol are broadlyactive as antileukemic agents in both myeloid and lymphoid diseases. Inconclusion, the data indicate that cytotoxic activity is not mediated bythe PML protein and therefore is not limited to diseases that areassociated with alterations in PML expression. Thus, the arseniccompounds of the invention have a potentially broader therapeutic rolethat is not confined to APL.

[0095] Clinical Study Of Melarsoprol In Patients With Advanced Leukemia

[0096] Melarsoprol, an organic arsenical synthesized by complexingmelarsen oxide with dimercaprol, has primarily been used for thetreatment of African trypanosomiasis. The effects of melarsoprol uponinduction of apoptosis in cell lines representative of chronic B-celllymphoproliferative disorders have been investigated, and the resultsare described below.

[0097] Melarsoprol (supplied as Arsobal [36 mg/mL] by Rhone PoulencRorer, Collegeville, Pa.) was diluted in propylene glycol at a stockconcentration of 10-4 mol/L and stored at room temperature. As₂O₃(Sigma, St. Louis, Mo.) was dissolved in 1.65 mol/L sodium hydroxide(NaOH) at a stock solution of 10-3 mol/L. Serial dilution (10⁻⁶ to 10⁻⁹mol/L) were made in RPMI 1640 media. An Epstein-Barr virus(EBV)-transformed B-prolymphocytic cell line (JVM-2), an EBV-transformedB-cell chronic lymphocytic leukemia (B-CLL) cell line (I83CLL), and onenon-EBV-transformed B-CLL cell line (WSU-CLL) were used as targets.Dose-response experiments with melarsoprol (10⁻⁷ to 10⁻⁹ mol/L) wereperformed over 96 hours.

[0098] Unexpectedly, the inventors found that melarsoprol caused a dose-and time-dependent inhibition of survival and growth in all three celllines. In contrast, As₂O₃ at similar concentrations had no effect oneither viability or growth. After 24 hours, all three cell lines treatedwith melarsoprol (10⁻⁷ mol/L) exhibited morphologic characteristics ofapoptosis. A prominent concentration-dependent downregulation of bcl-2mRNA after 24 hours of exposure to melarsoprol in WSU-CLL 183CLL, andJVM-2 cells was observed. Decrease of bcl-2 protein expression was alsoobserved in all three cell lines, whereas As₂O₃ had no effect on thisparameter.

[0099] Given that the in vitro data above have shown unexpectedly broadantileukemic activity for melarsoprol against both myeloid and lymphoidcells, and generally at lower concentrations than As₂O₃, a study wasinitiated to evaluate the pharmacokinetics, safety, and potentialefficacy of melarsoprol in human patients with relapsed or refractoryleukemia.

[0100] Eligible patients were treated with a brief IV injection dailyfor 3 days, repeated weekly for 3 weeks, with an additional 3 wk coursein responding pts. The initial dose was 1 mg/kg on Day 1, 2 mg/kg on Day2, and 3.6 mg/kg on Day 3 and all days thereafter. Parallel in vitrostudies included culture sensitivity of fresh leukemic cells to bothmelarsoprol and As₂O₃, along with serial flow cytometric studies ofsurface antigen expression, apoptosis, and bcl-2 expression. Threepatients with AML and one with CML have entered the study.

[0101] Using a method based on high performance liquid chromatographythat is sensitive to approximately 10 mg/ml, preliminary pharmacokineticdata show that peak plasma drug concentrations were obtained immediatelyafter injection with a Cmax that ranged from 1.2 ng/ml on day 1 to 2.4ng/ml on day 3. While the initial distribution phase was rapid, aprolonged Thy has suggested release from a deep compartment. Plasmaareas under the concentration×time curves (AUCs) were proportional tothe administered dose, ranging from 0.48 ng·hr/ml on Day 1 to 1.48ng·hr/ml on Day 3. Detectable concentrations of the drug were found inplasma one week after initial dosing. The drug has been relativelywell-tolerated. Adverse effects have included transient pain at theinjection site and mild nausea. No signs of “reactive encephalopathy”(occasionally observed during treatment of CNS trypanosomiasis) havebeen observed.

[0102] Results from these studies suggest that melarsoprol may havebroader activity than inorganic As₂O₃, and that concentrations which arecytotoxic to leukemic cells in vitro, and thus therapeutic, are readilyachieved in vivo.

[0103] Arsenic Trioxide Induces Apoptosis In K562 Chronic MyelogenousLeukemia (CML) Cells

[0104] A Philadelphia chromosome positive CML cell line K562 is used todetermine if arsenic trioxide (As₂O₃) promotes apoptosis in CML.Suspension cultures of cells in log phase were exposed to As₂O₃ atconcentrations of 1×10⁻⁵ M, 5×10⁻⁶ M, and 1×10⁻⁶ M. Aliquots of cellswere analyzed at various time points over the course of 72 hours toassess viability and apoptosis. Viability was measured using trypan blueexclusion; at the same time, apoptosis was detected by morphology, flowcytometry, and DNA gel electrophoresis.

[0105] Arsenic trioxide at a concentration of 1×10⁻⁶ M had no effect onK562 cell growth or viability. The greatest effect on cell growth andsurvival was seen with 1×10⁻⁵ M As₂O₃ K562 cell growth and viabilitydata after 72 hours of exposure to As₂O₃ are recorded in Table 1: TABLE1 % Cell Growth % Impairment Viability p value control 0 92.1 ± 0.9 5 ×10⁻⁶ M As₂O₃ 63.0 78.8 ± 0.5 0.0001 1 × 10⁻⁵ M As₂O₃ 75.3 61.9 ± 2.90.0223

[0106] Evidence that this arsenic-induced decrease in viabilityrepresented apoptosis was analyzed. Morphologic features of apoptosisincluding membrane blebbing and nuclear condensation were evident instained cytospins of K562 cells incubated with 10⁻⁵ M As₂O₃ for 72hours. This correlated with evidence of DNA internucleosomal damage asvisualized by gel electrophoresis of DNA extracted from K562 cellsexposed to 10⁻⁵ M As₂O₃. Quantitative assessment of apoptosis, asmeasured by the TUNEL method demonstrated that 75.6%±8.6 (1×10⁻⁵ MAs₂O₃) cells exhibited apoptosis as compared with 6.3%+±3.0 (control)cells at 72 hours. Treatment of K562 cells with 10⁻⁵ M As₂O₃ resulted inan upregulation of p21 mRNA, as detected by Northern analysis,suggesting an arrest of the cells in the G1 phase of the cell cycle.This data indicates arsenic trioxide as a therapeutic agent for CML.

[0107] Therapeutic Trials With Retinoic Acid And Arsenic Trioxide(As₂O₃) In PML-RARα AND PLZF-RARα Transgenic Mice

[0108] Acute promyelocytic leukemia (APL) is associated with chromosomaltranslocations which invariably involve the translocation of theRetinoic Acid Receptor α (RARα) locus on chromosome 17 to other loci inthe genome, such as in the majority of APL cases, the PML gene locatedon chromosome 15, and in a few cases the PLZF gene on chromosome 11.Patients harboring the t(15;17) are sensitive to treatment withAll-Trans Retinoic Acid (ATRA), yielding complete remission rates of 75%to 95%. APL associated with the t(11;17) (PLZF-RARα) shows a poorresponse to ATRA.

[0109] To test the efficacy of As₂O₃ in the treatment of APL, models ofthe disease were created in transgenic mice. Transgenic mice weregenerated by standard techniques in which the expression of the PML-RARαor PLZF-RARα fusion proteins is placed under the control of amyeloid-promyelocytic specific human Cathepsin-G (hCG) minigene. BothhCG-PML/RARα and hCG-PLZF-RARα transgenic mice develop myeloid leukemiawith features of APL similar to those in humans.

[0110] Therapeutic trials on these leukemic mice with the followingregimens were started: 1) ATRA: 1.5 μg per gram of body weight per dayadministered orally; and 2) ATRA: 6 μg per gram of body weight per dayadministered intraperitoneally. Mice were bled once a week to evaluatethe response.

[0111] PML/RARα leukemias responded well to ATRA with high remissionrates (80% with regimen 1). Surprisingly, in vitro, ATRA induceddifferentiation, and inhibited growth of leukemic cells as well asleukemic colony formation in bone marrow and spleen progenitors assaysin both PML-RARα and PLZF-RARα leukemias. Furthermore, in ex vivoexperiments, leukemic cells from PLZF-RARα mice lost their tumorigeniccapacity when transplanted in recipient nude mice upon pre-incubationwith ATRA, while untreated cells were tumorigenic. However, in vivo,PLZF-RARα leukemias responded poorly to ATRA (28% with regimen 1), whilehigher doses of ATRA appeared more effective (50% with regimen 2). Inconclusion, leukemias in PLZF-RARα transgenic mice are sensitive to ATRAtreatment, but might require therapeutic regimens with high doses ofATRA. These findings have direct implications in the treatment of APLpatients with t(11;17).

[0112] In both PML-RARα and PLZF-RARα leukemias, ATRA prolongedsurvival, but leukemias relapsed shortly after remission was achieved,and were refractory to further ATRA treatment. The two transgenic mousemodels is also used to test the efficacy and dosage of As₂O₃, andATRA+As₂O₃ in combination for treatment of APL patients resistant toATRA, and in APL associated with the t(11;17). A regimen of As₂O₃ at 6μg per day or a combination of As₂O₃ at 6 μg and ATRA at 1.5 or 6 μg pergram of body weight per day is administered intraperitoneally. Mice arebled weekly to evaluate the remission of the APL.

[0113] Manufacture and Stability Of Pharmaceutical Formulation

[0114] Solid ultrapure arsenic trioxide (As₂O₃) was solubilized in asolution of 5 M sodium hydroxide (NaOH). The suspension was stirred atambient temperature for 5 minutes which yielded a clear, homogenoussolution. The As₂O₃ solution (2 mL, 1.0 M) was added to 393.6 mL of H₂Oin a 500 ml Erlenmeyer flask, which yielded an As₂O₃ concentration of 1mg/mL at pH=12. A 5.0 M HCl solution was prepared by dilution of HCl(49.26 mL, 37% wt/wt, 10/15 M) with H₂O (50.74 mL) in a 250 mLErlenmeyer flask. The HCl solution was later transferred via syringe toa 1000 mL empty evacuated container. The As₂O₃ solution was backtitrated with HCl (0.725 mL, 5.0 M) to pH 8.0. Approximately 10 mL ofthe backtitrated As₂O₃ solution was filtered through a Millex-GS 0.22 μmfilter unit and was added to each of approximately 30 sterile evacuatedsterile vials. To make the pharmaceutical composition which would beinjected intravenously into patients, 10 mL of this solution waswithdrawn from two of the vials and was added to a 500 mL 5%-dextrosesolution which yielded a final pH of 6.5.

[0115] The high purity of the bulk starting material was confirmed (seeTable 1) by atomic absorptiometry. Duplicate samples of fourintermediate or final-step solutions were assayed for total arseniccontent. Assay bulk powder confirmed the extremely high purity of thestarting material. Data for arsenic content of the intermediate andfinished product solutions are presented in Table 2 below.

[0116] The data below show that the solutions are stable in that theredoes not appear to be any indication of weight loss of arsenic overtime. TABLE 2 Arsenic content (ppm) of intermediate formulation andfinished product solution of arsenic trioxide. Sample Code A-01* A-02A-03 A-04 A-05 Aliquot A 140,600 600 707 629 680 Aliquot B 139,000 564703 688 687 Assay Variance 1.1% 6% 0.57% 8.7%

[0117] Examples: Clinical Trials In Apl Patients

[0118] Arsenic trioxide was evaluated in patients with APL to determinewhether this agent induced either cytodifferentiation or apoptosis.Twelve patients who had relapsed from extensive prior therapy weretreated with arsenic trioxide at doses ranging from 0.06 to 0.2 mg/kgper day until a bone marrow remission was achieved. Bone marrowmononuclear cells were serially monitored by flow cytometry forimmunophenotype, fluorescence in situ hybridization (FISH), reversetranscription polymerase chain reaction (RT-PCR) assay for PML/RAR-αexpression, and Western blot expression of the apoptosis-associatedproteins, caspases 1, 2 and 3. The results showed that low-doses ofarsenic trioxide are highly effective for inducing complete remission inrelapsed patients with APL. Clinical response is associated withincomplete cytodifferentiation and induction of apoptosis with caspaseactivation in leukemic cells.

[0119] Methods

[0120] Clinical protocol: Eligibility requirements included a diagnosisof APL confirmed by cytogenetics or fluorescence in situ hybridization(FISH) analysis for a t(15;17) translocation, or by reversetranscriptase polymerase reaction (RT-PCR) assay for PML/RAR-α. Patientsmust have relapsed from standard therapy that had included all-transretinoic acid plus a combination of cytotoxic drugs. Signed informedconsent was required, and the protocol was reviewed and approved by thiscenter's institutional review board

[0121] Arsenic trioxide treatment: Arsenic trioxide was supplied as anaqueous solution in 10 ml vials containing 1 mg/ml of drug. The drug wasfurther diluted in 500 ml of 5%-dextrose solution and infusedintravenously over 2 to 4 hours once per day. While the initial cohortof patients received either 10 or 15 mg/day as a flat dose, the referralof two children prompted the invention of a weight-based regimen (0.15mg/kg/day) that was heretofore unknown. The drug was given daily untilbone marrow remission was observed. Patients who achieved completeremission were eligible for treatment with additional courses of therapy3 to 6 weeks after the preceding course. Subsequent courses weregenerally given at a dose of 0.15 mg/kg/day for a cumulative total of 25days, administered either daily or on a weekdays-only schedule, for amaximum total of 6 courses over approximately 10 months.

[0122] Monitoring during study: Patients with coagulopathy weretransfused with platelets and fresh-frozen plasma to maintain theplatelet count and fibrinogen at target levels ≧50,000 cells/cu mm and≧100 mg/dL, respectively. Blood counts, coagulation studies, serumchemistry profiles, urinalyses, and electrocardiograms were seriallyobtained. A bone marrow aspiration and/or biopsy was performed atbaseline and periodically thereafter until remission was documented.

[0123] Conventional response criteria were observed, which includedrecovery of bone marrow to ≦5% blasts, peripheral blood leukocytes≧3,000 cells/cu mm, and platelets ≧100,000 cells/cu mm.

[0124] Cellular immunophenotype studies: Heparinized bone marrow orblood samples were collected and mononuclear cells were isolated byFicoll-Hypaque centrifugation. Surface membrane antigens were detectedby direct immunofluorescence staining using fluorescein isethiocynate(FITC) or phycoerythrin conjugated monoclonal antibodies: CD16 (Leu111a), CD11b, CD33 (Leu M9). HLA-DR, CD45, and CD14, purchased fromeither Becton-Dickinson (Mountainview, Calif.) or Immunotech Immunology(Marseille, France). Dual-color staining was performed by incubatingcells simultaneously with two monoclonal antibodies, includingCD33-PE/CD11b-FITC and CD33-PE/CD16-FITC. Negative controls usingirrelevant monoclonal immunoglobulins of the same isotype were analyzedconcurrently. Flow cytometric analyses were performed on an EPICSProfile II flow cytometer (Coulter Electronics) equipped with a 488 nmargon laser. Forward and side-scatter cell parameters were measured andcombined with CD45/CD14 staining to identify populations of interest andto exclude monocytes from the analysis gate. The Multiparameter DataAcquisition and Display System (MDADS, Coulter Electronics) was used toacquire and analyze data.

[0125] Fluorescence in situ Hybridization (FISH)

[0126] Selected specimens that had undergone immunofluorescence stainingfor CD33 and CD11b were sorted for cells that coexpressed. both antigensusing a FACStar Plus cell-sorter (Becton-Dickinson). Separated cellswere incubated in culture media at 37° C. for one hour, treated withhypotonic solution 0.075M KCl for 5 minutes, fixed in 3:1methanol:acetic acid fixative, and air-dried. Interphase FISH wasperformed using a specific. PML/RAR-α translocation dual-color probe(Vysis; Downer's Grove, Ill.). Briefly, DNA from interphase cells wasdenatured by immersing slides in a solution of 50% formamide/2×SSC at73° C. for 5 minutes; the slides were then dehydrated in alcohol and airdried. A mixture of probe in hybridization mixture was applied, coveredwith a cover slip, and sealed with rubber cement. Hybridization wascarried out at 37° C. in a moist chamber for approximately 12 to 16hours. Following hybridization, unbound probe was removed by washing theslides at 45° C. in 50% formamide/2×SSC solution three times for 10minutes each, followed by a wash in 2×SSC/0.1 NP-40 solution at 45° C.for 5 minutes. Slides were then air dried and counter-stained with 4′,6-diamidino-2-phenylindole and covered with a glass coverslip. Analysisof interphase cells for fluorescent signals was performed with aPhotometrics Sensys camera fitted to a Zeiss axioscope. A minimum of 300cells was studied for each sample.

[0127] Western Blot Analysis

[0128] Cells were lysed in a buffer containing 50 mM Tris-HCl, 0.5 mMethylene glycol [bis]-[aminioacyl] tetra acetic acid, 170 mM NaCl, 1 mMdithiothreitol, 0.2% NP-40, 0.01 U/mL aprotinin, 10 μg/mL leupeptin, 10μg/mL pepstatin, and 1 AM phenylmethylsulfonyl fluoride (all fromSigma). The lysates were then sonicated using a ultrasonic homogenizer(4710 series, Cole Parmer Instruments, Chicago, Ill.) and centrifuged at7,500 g (Sorvall Instruments, Newtown, Conn.). Protein content of thelysates was determined using a BioRad Protein Assay Kit (Bio-RadLaboratories, Hercules, Calif.) at 595 nm with a BSA standard. A samplebuffer containing 10% glycerol, 0.4% SDS, 0.3% bromphenol blue, 0.2%pyronin Y, in 1× stacking buffer (Tris base 0.5 M, 0.8% SDS), 20%2-mercaptoethanol, was added to the cell lysates, which wereheat-denatured at 95° C. for 3 min. Subsequently, 15 μg/lane of proteinwas loaded on a SDS-polyacrylamkide gel containing 12.5% polyacrylamideand was size-fractionated by electrophoresis. Proteins wereelectroblotted onto Tras-Blot® transfer medium (Bio-Rad) and stainedwith Ponceau-S as an internal loading control. Rabbit anti-humanmonoclonal antibodies, including caspase 1, caspase 2 (both from SantaCruz Biotechnology, Santa Cruz, Calif.), and caspase 3 (PharMingen, SanDiego, Calif.) were added, and bound antibodies were detected using theECL™ chemiluminescence detect-ion system (Amersham, Arlington Heights,Ill.). Protein bands were quantified by computer densitometry.

[0129] RT-PCR Analysis for PML/RAR-α

[0130] RT-PCR was performed using methods previously described (Milleret al., 1992, Proc. Natl. Acad. Sci. 89:2694-8; Miller et al., 1993,Blood, 82:1689-94).

[0131] Results

[0132] Patients

[0133] Twelve patients with relapsed or refractory APL were treated. Allpatients had received extensive prior therapy with retinoids andcytotoxic drugs (Table 3). Two patients had relapsed from allogeneicbone marrow transplantation, one of whom had also failed donor T-cellreinfusion. One patient was being maintained on hemodialysis for chronicrenal failure.

[0134] Clinical Efficacy

[0135] Eleven of the 12 patients achieved a complete remission afterarsenic trioxide treatment. The patient who entered on hemodialysissustained an intracranial hemorrhage on day 1 and died on day 5. Themedian duration of therapy in responding patients was 33 days (range, 12to 39 days), the median daily dose was 0.16 mg/kg (range, 0.06 to 0.2mg/kg), and the median cumulative dose during induction was 360 mg(range, 160 to 515 mg) (Table 3). Complete remission by all criteria wasattained at a median time of 47 days (range, 24 to 83 days) afterinitiation of therapy. Remission by bone marrow criteri—the determiningfactor for discontinuing therapy—was achieved first, usually followed insequence by recovery of peripheral blood leukocytes and platelets. Overthe range of doses used in this study, no differences in efficacy ortime to response were obvious. After 2 courses of therapy, 8 of 11patients tested had converted their RT-PCR assays for PML/RAR-α frompositive to negative.

[0136] All 11 patients in complete remission completed at least 1post-remission treatment course with arsenic trioxide. Four, two, andone patient each have completed a total of three, four and fivetreatment courses, respectively. The median duration of remission is5+months (range, 1 to 9+months). However, 3 of the 11 patients relapsedduring the second treatment course; none of these patients had convertedtheir RT-PCR assays, and each appeared to have rapidly acquired drugresistance. Two of these individuals have since expired from progressiveleukemia.

[0137] Adverse Events

[0138] The clinical condition of patients in this study was highlyvariable, which reflected their extensive prior therapy. The protocoldid not require hospitalization; three patients completed inductiontherapy entirely as outpatients, and one other individual washospitalized solely for placement of a venous catheter. However, 8patients were hospitalized for complications of leukemia, 5 of whomrequired transfer to an intensive care unit, endotracheal intubation,and assisted ventilation for complications that included pulmonaryhemorrhage, renal failure, sepsis, graft vs. host disease, non-specificpulmonary infiltrates, or hypotension. One patient required insertion ofa permanent pacemaker after second-degree heart block developed in thesetting of severe metabolic acidosis, hyperkalemia, hypotension, andrenal insufficiency. However, the heart block reversed despiterechallenge with further arsenic trioxide therapy. The drug wastemporarily withheld due to serious intercurrent medical complicationsin 5 patients for a median of 2 days (range, 1 to 5 days). Two patientsdeveloped symptoms similar to that of the “retinoic acid syndrome”; bothwere presumptively treated with dexamethasone and improved. Only 2patients required no platelet transfusions whatsoever; the median numberof platelet units transfused was 61 (range, 0 to 586 units).

[0139] The median total peripheral blood leukocyte count at entry was4,700 cells/cu mm (range, 500 to 144,000 cells/cu mm). Six patientsdeveloped leukocytosis (i.e., ≧20,000 cells/cu mm) that ranged from20,800 to 144,200 cells/cu mm. No additional therapy was administered tothese patients, and the leukocytosis resolved in all cases withoutfurther intervention.

[0140] Common adverse reactions included lightheadedness during theinfusion, fatigue, musculoskeletal pain, and mild hyperglycemia. Threepatients developed dysesthias presumably due to peripheral neuropathy.However, 2 of these patients had been immobilized for prolonged periodsduring assisted ventilation, and the other patient had an antecedentneuropathic history.

[0141] Immunophenotype Studies

[0142] APL is characterized by cells that express CD33, an antigentypically associated with primitive myeloid cells. Arsenic trioxidetherapy induced a progressive decrease in the proportion of cells thatsolely expressed CD33, along with an increase in the proportion of cellsthat expressed CD11b, an antigen associated with mature myeloidelements. While these changes would be anticipated from any agent thatinduced remission in APL, arsenic trioxide also induced expression ofcells that simultaneously expressed both antigens. In most cases, thesedual-expressing cells dominated the myeloid cell population, and theypersisted for extended periods after complete remission was achieved byclinical criteria.

[0143] Fluorescence in situ Hybridization Analysis

[0144] Bone marrow mononuclear cells taken from a patient both early andlater in complete remission were sorted by flow cytometry forcoexpression of CD33 and CD11b. Using fluorescence in situ hybridization(FISH) analysis, three hundred cells were examined early in remission.Similar to control APL cells, the majority of these cells yielded ahybrid signal, indicating a translocation between PML and RAR-α genesand their origin from the neoplastic clone. However, when cells from thesame patient were again sorted using these same parameters later inremission, only the normal pattern of fluorescence signals was detected,indicating their derivation from normal hematopoietic progenitors.

[0145] Western Blot Analysis

[0146] Protein extracts from bone marrow mononuclear cells were seriallyexamined by Western blot analysis. The analysis showed that theprecursor forms of caspase 2 and caspase 3 were upregulated in vivo inresponse to arsenic trioxide treatment. Moreover, this treatment alsoinduced expression of cleaved fragments of caspase 1, indicatingactivation of the enzyme. There is also some indication that expressionof the cleaved form of caspase 3 is increased. The antibody used inthese experiments does not react with the cleaved form of caspase 2.

[0147] Discussion

[0148] In this study, with few exceptions, patients admitted to thetrial had sustained multiple relapses and were resistant to conventionalchemotherapy, retinoids, or bone marrow transplantation. At entry,patients in this study suffered from numerous leukemia-relatedcomplications, including respiratory failure, disseminated Varicellazoster infection, cavitary aspergillosis, chronic renal failure, andgraft-vs.-host disease. Moreover, 5 of the 12 patients requiredadmission to an intensive care unit for assisted ventilation andsupportive care, but these complications were not directly related toarsenic trioxide therapy.

[0149] Virtually all patients with a confirmed diagnosis of APL attainedremission without the early mortality associated with retinoid therapy.Although less commonly observed compared with all-trans retinoic acidtreatment, arsenic trioxide induced striking leukocytosis-in severalpatients. Upon withholding other cytotoxic drugs, the leukocytosisdisappeared as patients attained remission. Despite 3 early relapses, 8of 11 patients tested converted RT-PCR assays for PML/RAR-α (a molecularmarker of residual disease) to negative, a phenomenon that is unusualafter all-trans retinoic acid treatment alone. Finally, arsenic trioxideis active in APL over at least a three-fold dose range from 0.06 to 0.20mg/kg.

[0150] All-trans retinoic acid induces “terminal” differentiation of APLcells, but the cytodifferentiating effects of arsenic trioxide appear tobe incomplete. Arsenic induces a population of cells that simultaneouslyexpress surface antigens characteristic of both mature and immaturecells (i.e. CD11b and CD33, respectively). Early during induction, thesecells retain the t(15;17) translocation that characterizes APL.Unexpectedly, these cells persisted in the bone marrow despite theachievement of a clinically complete remission; however, later inremission, the coexpressing cells—while still readily detectable—were nolonger positive by in situ hybridization. The morphologic appearance ofleukemic cells during therapy is also far less distinctive than thatobserved during therapy with all-trans retinoic acid. In fact, leukemiccells from many patients displayed few morphologic changes for 10 ormore days, after which the proportion of leukemic cells progressivelydecreased.

[0151] Following “non-terminal” differentiation, arsenic trioxideappeared to induce apoptosis, coincident with increased expression andconversion of cysteine proteases (termed caspases) from inactiveprecursors to activated enzymes. The caspase pathway has only recentlybeen characterized as an important pathway of programmed cell death.Initially recognized due to homology between the C. elegans proteinced-3 and mammalian interleukin-1β converting enzyme (ICE), the familyof caspases now encompasses at least 10 different proteins that cleave anumber of polypeptides. In leukemic cell lines, caspase activation isinducible with a number of cytotoxic agents, including all-transretinoic acid. Since these enzymes induce widespread proteolysis, it isconceivable that PML/RAR-α is a caspase substrate.

[0152] A final similarity shared by arsenic trioxide and all-transretinoic acid is the rapid development of clinical resistance in someindividuals. Leukemic cells taken from two patients who relapsedretained in vitro sensitivity over concentrations ranging from 10−4 M to10⁻⁷ M. Relative arsenic resistance due to decreased intracellulartransport has been described in association with down-regulation ofmembrane transporters encoded by the ars operon in bacterial cells.

[0153] Resistance in mammalian cells is less well-characterized, butalterations in membrane transport or efflux are probably importantfactors.

[0154] In summary, arsenic trioxide induces complete remission inpatients with APL who have relapsed from extensive prior therapy. Thisdrug causes partial but incomplete cytodifferentiation of leukemiccells, followed by caspase activation and induction of apoptosis. TABLE3 Clinical characteristics and induction therapy results of patientswith acute promyelocytic leukemia treated with arsenic trioxide.Treatment Time To Platelets Leukocytes Age No. of duration Daily DoseCumulative Remission ≧100,000/ ≧3,000/ (yrs) Relapses (days) (mg/kg)Dose (mg) (days) cu mm cu mm 36 1* 36 .16 360 54 36 54 45 3*^(a) 39 .12390 83 39 83 31 3^(a,b) 37 .18 370 41 39 41 25 2 16 .06 160 24 16 16 622*^(d) 30 .11 300 41 41 31 75 1 12 .20 180 30 30 30 40 1* 33 .16 495 4747 43 13 2*^(a,b) 27 .18 270 50 41 52 9 1* 33 .17 165 28 28 28 70 1^(c)28 .16 420 77 77 49 28 2* 36 .15 515 54 47 54 25 3 5 .15 75 † † †

[0155] All patients had previously received one or more courses ofall-trans retinoic acid, plus an anthracycline antibiotic plus cytosinearabinoside. ★ Denotes individuals with proven retinoid resistance (i.e.lack of response during reinduction or relapse while on retinoidmaintenances; † Denotes patient who died early. Other treatment: ^(a)mitoxantrone/etoposide; ^(b) allogeneic bone marrow transplantation;^(c) methotrexate/vincristine/6-mercaptopurine; ^(d) 9-cis retinoic acidplus M195 (anti-CD33 monoclonal antibody).

[0156] Examples Clinical Use In Lymphoma

[0157] Based upon the initial discovery of the antitumor effects ofarsenic trioxide in vitro against B-cell lymphocyte lines, the inventorstreated one patient with intermediate-grade large cell lymphoma who hadrelapsed from multiple forms of conventional therapy, includingautologous bone marrow transplantation. Despite rapid progression of hisdisease prior to starting the arsenic trioxide therapy, treatment witharsenic trioxide effected a major (>50%) shrinkage in the size of hiscancerous lymph nodes and spleen, which was also associated with a majorimprovement of his quality of life.

[0158] Examples

[0159] Clinical Use In Non-Hematopoietic Cancer

[0160] Arsenic trioxide was also used to treat colon cancer. In apreliminary test, one patient with colon cancer who received onetreatment with arsenic trioxide showed a major reduction in his serumCEA (carcinoembryonic antigen) level. The patient received dailyintravenous infusion of 0.1-5 mg arsenic trioxide per kg body weigh perday for five days. A change in the level of CEA from 19,901 ng/ml to15,266 ng/ml, a 23% reduction, was observed. It is well known that the areduced level of serum CEA is associated with antitumor response.

[0161] Clinical data confirms that arsenic trixoide can also be used totreat other non-hematopoietic cancer, such as colon cancer.

[0162] Examples Pharmacokinetics Studies

[0163] Several dose-ranging studies were conducted to examine thepharmacokinetics (PK) and biological effects of As₂O₃ in patients withAPL and in patients with other hematologic diseases. In patients withAPL, marrow mononuclear cells were serially monitored by flow cytometryfor immunophenotype, fluorescence in situ hybridization (FISH), andWestern blot expression of the apoptosis-associated proteins, caspases1, 2 and 3. Cells that coexpressed CD11b and CD33, and which by FISHanalysis carried the t(15;17) translocation, progressively increasedduring treatment and persisted early in complete remission. As₂O₃ alsoinduced in vivo expression of the proenzymes of caspase 2 and caspase 3,and activation of both caspase 1 and caspase 3. PK analysis of blood andurine for elemental arsenic (As) content showed that As was distributedin both plasma and red blood cell fractions of whole blood. Parallelelimination curves suggested that these 2 compartments were freelyexchangeable, and decayed from peak values with initial half lives ofabout 60 mins. The mean AUC on day 1 was about 400 ng·hr/ml.Approximately 20% of the administered dose was recovered in urine withinthe first 24 hrs.

[0164] We then initiated a dose-ranging study in patients with diseasesother than APL using a daily intravenous dosing schedule for acumulative total of 25 days per treatment course every 3-5 weeks at doselevels of 0.1 and 0.15 mg per kg body weight per day. To date, 10patients have been accrued, including patients with CLL (2 patients),AML (3 patients), lymphoma (4 patients), and CML (1 patient). Fivepatients were removed from the study early due to rapid progression, and5 completed the planned 25-day course. Over this dose range, the drughas proved well-tolerated; adverse effects have included skin rash,lightheadedness during the infusion, fatigue, and QTc prolongation onEKG. Results from this ongoing study show that clinical use of As₂O₃induces partial differentiation and apoptosis in APL, but that thetherapeutic effects of this agent are not confined to this disorder.

[0165] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0166] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1. A kit, comprising arsenic trioxide and instructions for theadministration of said arsenic trioxide to a patient diagnosed withacute promyelocytic leukemia, comprising delivering a therapeuticallyeffective dosage amount of about 0.15 mg/kg of said arsenic trioxide fora maximum of 60 days or until bone marrow remission, which constitutes afirst administration.
 2. The kit of claim 1, further comprisinginstructions for a second administration of said arsenic trioxide,wherein said second administration comprises a therapeutically effectivedosage amount of about 0.15 mg/kg of arsenic trioxide for 25 doses. 3.The kit of claim 2, wherein further comprising instructions wherein saidsecond administration is administered 3 to 6 weeks after said firstadministration.
 4. The kit of claim 3, further comprising instructionswherein said second administration is administered for up to five weeks.5. The kit of claim 4, further comprising instructions wherein saidsecond administration is administered at five doses per week.
 6. The kitof claim 2, further comprising instructions for repeating said secondadministration.
 7. The kit of claim 6, further comprising instructionswherein said second administration is repeated every 3 to 6 weeks. 8.The kit of claim 7, further comprising instructions wherein said secondadministration is repeated until a total of between two and ten cyclesof said second administration are completed.
 9. The kit of claim 8,further comprising instructions for repeating said second administrationuntil a total of two cycles of said second administration are completed.10. The kit of claim 8, further comprising instructions for repeatingsaid second administration until a total of ten cycles of said secondadministration are completed.
 11. A kit, comprising arsenic trioxide andinstructions for the administration of said arsenic trioxide to apatient diagnosed with acute promyelocytic leukemia, comprisingdelivering a therapeutically effective dosage amount of about 0.15 ofsaid arsenic trioxide once a day for a maximum of 60 days or until bonemarrow remission, which constitutes a first administration.
 12. The kitof claim 11, further comprising instructions for a second administrationof said arsenic trioxide, wherein said second administration comprises atherapeutically effective dosage amount of about 0.15 mg/kg of arsenictrioxide once a day for 25 doses.
 13. The kit of claim 12, whereinfurther comprising instructions wherein said second administration isadministered 3 to 6 weeks after said first administration.
 14. The kitof claim 13, further comprising instructions wherein said secondadministration is administered for up to five weeks.
 15. The kit ofclaim 14, further comprising instructions wherein said secondadministration is administered at five doses per week.
 16. The kit ofclaim 12, further comprising instructions for repeating said secondadministration.
 17. The kit of claim 16, further comprising instructionswherein said second administration is repeated every 3 to 6 weeks. 18.The kit of claim 17, further comprising instructions wherein said secondadministration is repeated until a total of between two and ten cyclesof said second administration are completed.
 19. The kit of claim 18,further comprising instructions for repeating said second administrationuntil a total of two cycles of said second administration are completed.20. The kit of claim 18, further comprising instructions for repeatingsaid second administration until a total of ten cycles of said secondadministration are completed.